Many crop species produce seed oils in which the fatty acid composition is not ideally suited to the intended use. The application of conventional breeding methods, coupled in some cases with mutagenesis, has resulted in the production of new varieties of several species with desirable alterations in the fatty acid composition of seed oil. A notable example is the development of low erucic acid varieties of rapeseed (Stefansson 1983). Similar efforts have resulted in the reduction of the level of polyunsaturated 18-carbon fatty acids in soybean (Wilcox and Cavins 1985; Graef et al. 1988), sunflower (Fick 1989), and linseed oils (Green and Marshal 1984).
Most of the genetic variation in seed lipid fatty acid composition appears to involve the presence of an allele of a gene that disrupts normal fatty acid metabolism and leads to an accumulation of intermediate fatty acid products in the seed storage lipids (Downey 1987). However, it seems likely that, because of the inherent limitations of this approach, many other desirable changes in seed oil fatty acid composition may require the directed application of genetic engineering methods.
α-Linolenic acid (18:3Δ9,12,15) is an eighteen carbon fatty acid containing three cis double bonds at the 9-10, 12-13 and 15-16 carbons. It is found in the cells of higher plants as a constituent of cell membranes. It is also found in storage organs, such as in seeds. There it is designated oil bodies which are bounded by an electron dense structure that is thought to be a half-unit membrane and dispersed in the cytoplasmic environment of cells. When present as a constituent of cell membranes, linolenic acid is usually esterified to the sn-1 or sn-2 position of the glycerol moiety of a diacyl-glycerolipid. By contrast, when present in oil bodies, linolenic acid is usually esterified to the sn-1, sn-2 or sn-3 position of a triacylglycerolipid (TAG).
Linolenic acid is extensively used in the paint and varnish industry in view of its rapid oxidation. Flax seed is a predominant source of this oil. Soybean seed, on the other hand, does not have sufficient linolenic acid content to be used in this industry. Thus, increasing the linolenic acid content in a plant such as soybean would permit the use of the soybean oil in the paint and varnish industry.
On the other hand, it is undesirable to have significant levels of linolenic acid in cooking oils and foods. Linolenic acid is unstable during cooking and is rapidly oxidized. The oxidized products impart rancidity to the finished product. A rapeseed or soybean oil with reduced linolenic acid, such as containing 2% or less of linolenic acid, would be ideal for use as a cooking oil.
Linolenic acid is also a precursor in the biosynthesis of jasmonic acid, an important plant growth regulator. Linolenic acid is converted to jasmonic acid by introduction of an oxygen to the carbon chain by a lipoxygenase, followed by dehydration, reduction, and several β-oxidations (Vick and Zimmerman, 1984). The activity of jasmonic acid has been measured in terms of induction of pathogen defense responses. By application of free linolenic acid to plants, plant pathogen defenses can also be induced (Farmer and Ryan, 1992).
A model has been proposed to explain the ability of free linolenic acid to exhibit the effects associated with jasmonic acid (Farmer and Ryan, 1992). It is hypothesized that all of the enzymatic activities which are required for the conversion of linolenic acid to jasmonic acid are constitutively present in the cell and the rate limiting step in the production of jasmonic acid is the availability of free linolenic acid. A likely route for the production of the free linolenic acid is by the activity of a lipase in the plasma membrane.
It has been observed that exogenous jasmonic acid can more powerfully activate defense responses than can wounding. This suggests that wounds cannot generate enough free linolenic acid to support high level production of jasmonic acid. The activity of the lipase or the availability of appropriate substrate for the lipase may be rate limiting upon wounding. Thus, increasing the linolenic acid content of plasma membrane may positively influence “signal transduction” in plants and result in better protection against environment and pathogen stress.
Linolenic acid, as well as oleic and linoleic acids are also important constituents, as well as precursors of volatile carbonyl compounds, whic contribute to the aroma of both fresh and cooked foods. The major fatty acids of tomato fruit pericarp are oleic, linoleic and linolenic acids. As the fruit ripens, the levels of the latter two fatty acids decline resulting in the production of a number of 4-6 carbon containing aldehydees and ketones. One particular metabolite, cis-3-hexanol, has been shown to be present in higher levels in vine-ripened tomatoes compared to supermarket tomatoes or tomatoes stored in refrigerators. It is likely, therefore, that the “aroma” of fresh fruits and vegetables can be “modulated” by regulation of the content of linolenic and linoleic acids, important substrates for the enzyme lipoxygenase and subsequently the hydroperoxide cleaving enzyme, which generates the volatile “aroma” compounds.
From the above, it is clear that the ability to vary the content of linolenic acid in plants would be desirable. However, to achieve this result it is necessary to determine what controls the product of linolenic acid in plants.
A large body of experimental evidence derived from radiochemical tracer studies has indicated that α-linolenic acid is synthesized by the desaturation of linoleic acid (18:2Δ9,12) (reviewed in Harwood 1988;). However, the actual substrate for desaturation is not known.
In vivo and in vitro labelling studies suggest that there are possibly two distinct pathways for the synthesis of linolenic acid (Browse and Somerville, 1991). One possible pathway is thought to be located in the endoplasmic reticulum where linoleic acid esterified to the sn-2 position of phosphatidylcholine is a substrate for desaturation. However, the available evidence does not exclude the possibility that linoleic acid esterified to other lipids may also be a substrate.
A second possible pathway of linoleic acid desaturation is located in the plastid where the available evidence suggests that linoleic acid esterified to monogalactosyldiacylglycerol and, possibly, other plastid lipids is the substrate for desaturation.
Relatively little direct information is available concerning the enzymes involved in linoleic acid desaturation. Low levels of enzyme activity have been detected in microsomal membrane preparations from developing linseed (Linum ussitatum) (Browse and Slack, 1981) and, more recently, in preparations of gently lysed chloroplasts (Schmidt and Heinz, 1990a,b). The general features of the enzyme may be inferred from information available about other enzymes of this class.
The most thoroughly characterized desaturase is the stearoyl-Coenzyme A (CoA) desaturase from vertebrate liver (reviewed by Holloway, 1983). This enzyme has been shown to be an integral membrane protein which contains non-heme iron. The desaturase reaction requires fatty acyl-CoA, molecular oxygen and reduced cytochrome b5, another membrane protein. In vivo, the reduced cytochrome b5 is produced by the transfer of reducing equivalents from NADH via the activity of cytochrome b5 reductase, a flavin containing membrane protein.
The most thoroughly characterized desaturase from plants is the stearoyl-ACP desaturase (McKeon and Stumpf, 1982; Shanklin and Somerville, 1991). This enzyme also requires molecular oxygen and a high potential reductant. However, in contrast to the animal enzyme, this desaturase is a soluble plastid protein which preferentially acts on a fatty acid esterified to acyl carrier protein (ACP) rather than CoA. This enzyme also differs from the animal enzyme by utilizing reduced ferredoxin as an intermediate electron donor.
Other plant desaturases appear to be membrane proteins. The microsomal Δ12 oleate desaturase from several plant species has been assayed in membrane preparations from several plants (Harwood, 1988). As with the stearoyl-CoA desaturase from animals, this enzyme requires molecular oxygen and reduced cytochrome b5 as an electron donor (Kearns et al., 1991). However, it appears that oleate esterified to a phospholipid is the substrate rather than a CoA ester.
With regard to the activity responsible for the making of linolenic acid, little was known as to its source or origin. However, evidence that the amount of linolenic acid is related to the amount of linoleic acid desaturase activity has been obtained by analysis of the properties of the fad3 mutant of Arabidopsis thaliana (Lemieux et al. 1990). This mutant is deficient in linolenic acid in the storage oils of its seed lipids and in the membrane lipids of different tissues to varying degrees. The mutant also had an increase in the amount of linoleic acid. This can be interpreted as evidence that the mutant is defective in the activity of a desaturase which converts linoleic acid to linolenic acid.
There is further evidence to suggest that the activity of this desaturase could be rate limiting for linolenic acid synthesis under normal circumstances. This was discovered by measuring the effects on fatty acid composition in heterozygous plants (i.e., fad3+/fad−) formed by crossing the wild type with the fad3 mutant. In these F1 plants, which have one copy of the normal fad3 gene product instead of the two normally found in the wild type, the amount of linolenic acid was almost exactly intermediate between that found in either parent. This suggests that the amount of linolenic acid is proportional to the amount of functional fad3 gene product (Lemieux et al., 1990).
These results do not shed any light, however, on the nature of the fad3 gene product or whether the observed effects in mutants are related to either a decrease in quantitiy of desaturase protein or desaturase activity due to a defective protein.
Moreover, nothing is known with any degree of certainty about the linoleic acid desaturase from plant microsomes. As noted above, very little is known about the microsomal desaturases except that they probably utilize reduced cytochrome b5 as intermediate electron donor and probably utilize lipids rather than CoA or ACP esters as substrates.
Moreover, as in many other aspects of plant biology, the lack of specific information about the biochemistry and regulation of lipid metabolism makes it difficult to predict how the introduction of one or a few genes might usefully alter seed lipid synthesis.
An additional problem arises from the fact that many of the key enzymes of lipid metabolism are membrane-bound and present in low quantities. Thus, attempts to solubilize and purify them from plant sources have not been successful.